CN110818811B - Method for extracting and separating high-viscosity narcissus polysaccharide from narcissus bulb - Google Patents
Method for extracting and separating high-viscosity narcissus polysaccharide from narcissus bulb Download PDFInfo
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Abstract
The invention belongs to the technical field of extraction and separation of active ingredients of natural plants, and particularly relates to a method for extracting and separating high-viscosity narcissus polysaccharide from narcissus bulbs; narcissus bulb can secrete a mucilage when physically damaged by insects such as gnawing and the like, and the main component of the mucilage is macromolecular polysaccharide-narcissus polysaccharide; the invention provides a method for extracting and separating narcissus polysaccharide, which has high viscosity and no starch and alkaloid residue.
Description
Technical Field
The invention belongs to the technical field of natural medicinal chemistry, relates to a natural plant active ingredient extraction and separation technology, and particularly relates to a method for extracting and separating high-viscosity narcissus polysaccharide from narcissus bulbs.
Background
Narcissus (Narcissus tazetta L.) is a perennial root herbaceous plant of Narcissus of Amaryllidaceae, has fragrant and elegant flower and high ornamental value, and is one of ten traditional famous flowers in China. The flower of the various flowers is planted in the places of Fujian, Jiang Zhe and the like in China, wherein the flower of the various flowers in Zhangzhou is famous and the planting area is the largest. According to the literature report [1], the narcissus bulb contains various bioactive chemical substances, such as alkaloid compounds including narcissus and pseudolycorine, saccharide substances including glucose, mannose, starch, mucopolysaccharide, cellulose and the like, and flavonoid compounds including narcissus and rutin. Among them, alkaloids have been receiving general attention because of their anticancer activity, while mucopolysaccharides have been less studied. Experiments show that the viscous polysaccharide has the properties of good wire drawing, specific shear thinning, good thickening and the like, and has potential application value in the fields of cosmetics and the like.
Patent 2010105077001[1]Provides a Chinese narcissus ballThe method for extracting and separating narcissus polysaccharide from stems adopts a hot water extraction method to extract the narcissus corm polysaccharide, which can cause starch in the narcissus corm to be simultaneously extracted along with the polysaccharide and to be gelatinized, thereby causing the prepared narcissus polysaccharide to contain a large amount of gelatinized starch, and the subsequent purification is quite difficult. Literature reference[3,4]A process for extracting the narcissus polyose from the bulb of narcissus is disclosed, which includes reflux defatting with alcohol/petroleum ether, freeze drying, pulverizing and extracting in hot water. Like the above patents, the hot water extraction easily causes the prepared narcissus polysaccharide to contain a large amount of gelatinized starch, and the cost is very high by adopting freeze-drying. In addition, another disadvantage of using hot water extraction is that it is liable to cause the narcissus polysaccharide to degrade, resulting in a severe reduction in the viscosity of the product obtained.
The processes provided by the above patents or documents have the following problems: (1) the powder can be crushed only by degreasing with ethanol or petroleum ether and then vacuum drying or freeze drying, so that the process is complex and time-consuming, and the production cost is high; (2) hot water extraction is adopted to cause starch gelatinization and difficult separation from polysaccharide; (3) the temperature of hot water used in leaching is high (80-100 ℃), and polysaccharide is easy to degrade.
Disclosure of Invention
In order to solve the problems, the invention provides a method for extracting and separating high-viscosity narcissus polysaccharide from narcissus bulbs.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a method for extracting and separating high-viscosity narcissus polysaccharide from narcissus bulbs comprises the following steps:
(1) cleaning: taking fresh narcissus bulb, peeling off the outer skin, cutting off the root hair, and washing with clear water;
(2) crushing or homogenizing: pouring the cleaned narcissus bulb and the aqueous solution containing the preservative and the hydrolase inhibitor into a crusher for pulping;
(3) leaching: adding an aqueous solution containing a preservative and a hydrolase inhibitor into the homogenate and continuously stirring;
(4) coarse filtration: filtering the leaching solution with filter cloth;
(5) adjusting the pH value: adjusting the pH value to weak acidity by using acid, and uniformly stirring;
(6) centrifuging: respectively collecting supernatant and bottom precipitate;
(7) and (3) separating the lectin: freeze drying the precipitate to obtain crude lectin;
(8) concentration: concentrating the supernatant in vacuum;
(9) alcohol precipitation: precipitating the concentrated solution with ethanol, and rinsing the ethanol precipitate with ethanol water;
(10) and (3) drying: and (5) drying the alcohol precipitate in vacuum to obtain the narcissus polysaccharide.
Further, the raw material crushing and homogenizing mode in the step (2) comprises homogenizing, extruding and shredding.
Further, the aqueous leaching solution used in the steps (2) and (3) contains a preservative and a hydrolase inhibitor; the leaching temperature is 0-50 ℃; the leaching time is 0-4 h; stirring is needed during leaching, and the stirring speed is 0-120 r/min.
Further, the preservative used in the steps (2) and (3) is potassium sorbate; the addition amount is 0-1%.
Further, the hydrolase inhibitor used in the steps (2) and (3) is a metal ion complexing agent, including lichenate, citrate, phytic acid and the like, preferably disodium lichenate; the addition amount is 0-5%, preferably 0.1%;
further, the weak acidity in the step (5) is 2-6; the reagents used are acidic chemical reagents.
Compared with the polysaccharide extraction process in the prior art, the process provided by the invention has the following characteristics:
(1) the viscous liquid (namely the polysaccharide extracted by the invention) secreted by the narcissus bulb is mainly stored in the intercellular layer, is a mechanism for resisting insect gnawing and microbial infection of plants, and can be automatically secreted when the skin of the narcissus bulb is damaged. Therefore, the narcissus bulb is only required to be fully crushed, the viscous liquid is quickly secreted and dissolved in water, and heating or ultrasonic-assisted extraction is not required.
(2) Generally, plant tissue slurry is rich in various nutrients, and microorganisms can easily reproduce at room temperature. Therefore, a suitable amount of preservative is added to the homogenate/extract.
(3) The plant vacuole is rich in various hydrolytic enzymes, can hydrolyze the narcissus polysaccharide, seriously reduces the viscosity of the narcissus polysaccharide, and therefore must be inactivated. The mainstream method for enzyme inactivation is heating inactivation, but the high temperature can cause the degradation of the narcissus polysaccharide. In the other method, a chemical reagent inhibits enzyme activity, and experiments show that EDTA and the like have good enzyme inhibition effect and can effectively keep the viscosity of the leaching solution.
(4) Experiments show that the narcissus agglutinin is easy to precipitate at the pH of 3-4, and is dissolved when the pH is continuously reduced, so that the isoelectric point of the narcissus agglutinin is preliminarily judged to be at the pH of 3-4, and the narcissus agglutinin is separated by adopting a method of combining the isoelectric point with centrifugation.
Description of the drawings:
FIG. 1 is a process flow diagram;
figure 20.5% narcissus polysaccharide viscometry results;
FIG. 3 is an HPLC chromatogram of a narcissus polysaccharide sample;
FIG. 4 is an HPLC chromatogram of a narcissus bulb sample;
figure 5 is a graph showing the inhibitory effect of narcissus polysaccharides on its histamine release.
Detailed Description
The present invention is further illustrated by the accompanying fig. 1-5 and the detailed description, which describe only some embodiments of the invention and are not intended to limit the scope of the invention. Other embodiments, which can be made by persons skilled in the art without any inventive contribution, fall within the scope of protection of the present invention.
A method for extracting and separating high-viscosity narcissus polysaccharide from narcissus bulbs comprises the following steps:
(1) cleaning: taking fresh narcissus bulb, peeling off the outer skin, cutting off the root hair, and washing with clear water;
(2) crushing or homogenizing: pouring the cleaned narcissus bulb and the aqueous solution containing the preservative and the hydrolase inhibitor into a crusher for pulping;
(3) leaching: adding an aqueous solution containing a preservative and a hydrolase inhibitor into the homogenate and continuously stirring;
(4) coarse filtration: filtering the leaching solution with filter cloth;
(5) adjusting the pH value: adjusting the pH value to weak acidity by using acid, and uniformly stirring;
(6) centrifuging: respectively collecting supernatant and bottom precipitate;
(7) and (3) separating the lectin: freeze drying the precipitate to obtain crude lectin;
(8) concentration: concentrating the supernatant in vacuum;
(9) alcohol precipitation: precipitating the concentrated solution with ethanol, and rinsing the ethanol precipitate with ethanol water;
(10) and (3) drying: and (5) drying the alcohol precipitate in vacuum to obtain the narcissus polysaccharide.
The above-described manner is explained in detail below with reference to several embodiments:
the first embodiment is as follows: preparation of narcissus bulb polysaccharide
(1) Taking fresh narcissus bulb, peeling off the outer skin, cutting off the root hair, and washing with clear water;
(2) adding 200g of water hyacinth bulbs into 400mL of aqueous solution containing 0.05 percent of potassium sorbate and 0.1 percent of EDTA, and pulping by using a homogenizer;
(3) supplementing 1600mL of aqueous solution containing 0.05% of potassium sorbate and 0.1% of EDTA into the homogenate, and slowly stirring for 30 min;
(4) filtering with 200 mesh nylon cloth, and collecting filtrate;
(5) adjusting the pH value to 3-4 by trichloroacetic acid, and stirring for 30min at a speed of 15 r/min;
(6) centrifuging at 2000G for 30min, and collecting supernatant;
(7) adjusting the pH of the supernatant to about 6 with alkali, and concentrating the supernatant to 3-5 degrees brix by using a rotary evaporator;
(8) adding 3 times of alcohol into the concentrated solution, slowly stirring, standing for 60min, filtering with 100 mesh nylon cloth, collecting precipitate, and rinsing the precipitate with 70% alcohol for 2 times;
(9) the precipitate is dried in vacuum, and 8.21g of narcissus polysaccharide is obtained.
Example two: preparation of narcissus bulb polysaccharide
(1) Taking fresh narcissus bulb, peeling off the outer skin, cutting off the root hair, and washing with clear water;
(2) shredding 2kg of water clematis bulbs, and putting into an extraction tank;
(3) adding 10kg of aqueous solution containing 0.05% potassium sorbate and 0.1% EDTA into the leaching tank, stirring for 120min, and discharging; leaching for 2 times again, and the solvent is 5kg each time;
(4) mixing the leaching solutions, filtering with 200 mesh nylon cloth, and collecting filtrate;
(5) adjusting the pH value to 3-4 by trichloroacetic acid, and stirring for 30min at a speed of 30 r/min;
(6) centrifuging by using a tubular centrifuge, and collecting a light phase;
(7) adjusting the pH value of the light phase to about 6 by using alkali, and concentrating the light phase to 3-5-degree brix by using a falling film evaporator;
(8) adding 3 times of alcohol into the concentrated solution, slowly stirring for 90min, filtering with 100 mesh nylon cloth, collecting precipitate, and rinsing the precipitate with 70% alcohol for 2 times;
(9) the precipitate is dried in vacuum, and 76.59g of narcissus polysaccharide is obtained.
Example three: preparation of narcissus bulb polysaccharide
(1) Taking fresh narcissus bulb, peeling off the outer skin, cutting off the root hair, and washing with clear water;
(2) putting 100kg of water hyacinth bulbs into an extraction tank after passing through an extruder;
(3) adding 1000kg of aqueous solution containing 0.05% potassium sorbate and 0.1% EDTA into the leaching tank, stirring for 120min, and discharging; leaching for another time for 1 time, wherein the solvent is 800 kg;
(4) mixing the leaching liquor, filtering by using a filter bag with 200-400 meshes, and collecting filtrate;
(5) adjusting the pH value to 3-4 by using citric acid, and stirring for 30min at the speed of 60 r/min;
(6) centrifuging by using a tubular centrifuge, and collecting a light phase;
(7) adjusting the pH value of the light phase to about 6 by using alkali, and concentrating the light phase to 3-5-degree brix by using a falling film evaporator;
(8) adding 2.5 times of alcohol into the concentrated solution, slowly stirring for 120min, filtering with 100 mesh nylon cloth, collecting precipitate, and rinsing the precipitate with 75% alcohol for 2 times;
(9) the precipitate is dried in vacuum, namely 3.7kg of narcissus polysaccharide.
To demonstrate the effectiveness of the present invention, the following experiments were performed:
test of physicochemical Properties of Narcissus (one) polysaccharide
1. Viscosity measurement of narcissus polysaccharide
The main reagents are as follows: 0.5% Narcissus polysaccharide (example three)
The method comprises the following steps: selecting a viscosity-shearing project (the shearing force range is 0.1-10 s) by using an Antopa rheometer MCR92 at 25 DEG C-1) And (4) carrying out measurement.
The result is shown in figure 2, the average viscosity of the narcissus polysaccharide solution is 40 mPas, the viscosity of the narcissus polysaccharide solution firstly slightly rises with the increase of the shearing force and then rapidly falls, the narcissus polysaccharide solution has obvious shear thinning, and the narcissus polysaccharide solution is homogenized or emulsified by using the feed liquid.
2. Narcissus polysaccharide starch residue detection
The main reagents are as follows: 2.5% of I2KI solution (2.5 gI)2+5.0gKI, add water to 100 mL); 2.0% narcissus polysaccharide solution (2.0 g narcissus polysaccharide (example one) is taken, water is added to 100mL, the mixture is heated to boil, and the mixture is naturally cooled to room temperature); 0.2% Narcissus polysaccharide solution (0.2 g Narcissus polysaccharide was taken (according to the reference)[2]Extracting), adding water to 100mL, heating to boil, and naturally cooling to room temperature); 0.005% starch (50 mg of starch was added to 1000mL of water, heated to boiling, and cooled to room temperature).
The identification method comprises the following steps: respectively taking 5mL of samples to be detected in 25mL colorimetric tubes, and adding 0.1mLI2KI solution, shaking up and observing color change.
The test results are shown in Table 1, and the narcissus polysaccharide sample prepared by the method of the invention is processed by I2the/KI test shows negative, i.e. the starch content is below 0.25%; narcissus polysaccharide sample prepared in reference 2, preparation I2The KI test shows positive, and the residual amount of starch is about 3 percent by colorimetric estimation.
TABLE 1 starch test results
3. Narcissus polysaccharide alkaloid substance residue detection
Sample treatment: (1) pulping 10g of Narcissus bulb, extracting with 70% ethanol at 60 deg.C for 2 times, mixing extractive solutions, diluting to 100.0mL with 70% ethanol, filtering the filtrate with 0.45 μm microporous filter head, and analyzing the filtrate with liquid chromatography. (2) Taking 10.0g narcissus polysaccharide (example 2), pulverizing, extracting with 70% ethanol at 60 deg.C for 2 times, mixing extractive solutions, adding 70% ethanol to constant volume to 100.0mL, filtering the filtrate with 0.45 μm microporous filter head, and analyzing the filtrate by liquid chromatography.
Chromatographic conditions are as follows: shimadzu LC-16, autosampler SIL-16, Colun Oven CTO-16L column Oven, detector SPD-M20A, chromatographic column (Wondasil C18-WR 5 μ M4.6 × 150mm), sample injection volume 5 μ L; mobile phase: 10% methanol → 100% methanol (35 min); detection wavelength: 225 nm.
The detection results are shown in fig. 3 and 4. FIG. 3 shows that the narcissus polysaccharide sample prepared in the example has no peak except the solvent peak and around 4.5min and 10min, while the alkaloid in Amaryllidaceae generally has multiple conjugated double bonds (chromophoric groups) and can be detected by an ultraviolet detector, which indicates that the sample contains almost no alkaloid. And 8 peaks appear in the narcissus bulb sample at 10-20 min, wherein the peak (Rt is 15.5min) is colchicine, and the content of the colchicine is 0.23% calculated according to a peak area external standard method. In conclusion, the narcissus polysaccharide preparation process provided by the invention can effectively remove the mid-colchicine in the raw materials and avoid influencing the safety of the narcissus polysaccharide due to the residue of alkaloid.
Biological activity test of narcissus polysaccharide
1. Moisture retention efficacy
Experimental samples: pure water, 0.5% of glycerol, 0.5% of hyaluronic acid, 0.5% of beta-glucan and 0.5% of narcissus polysaccharide;
the test method comprises the following steps: the test was carried out in an environment of 40% humidity and 25 ℃. Dividing the inner skin of arm of subject into 5 pieces (2.5CM × 4CM), smearing 0.2mL of above solution, measuring water change with Corneometer CM825 probe of multifunctional skin tester (CK-MPA10) after 60min, taking data three times, and averaging.
The test results are shown in table 2, and the test results show that the narcissus polysaccharide has a good moisturizing effect, and the moisturizing effect is equivalent to that of hyaluronic acid.
TABLE 2 evaluation of moisturizing Effect
2. Anti-allergy effect
The RBL-2H3 cell model is used to explore the inhibitory effect of narcissus polysaccharide on histamine release. RBL-2H3 cells were sensitized with IgE (0.2. mu.g/mL) and stimulated with DNP-BSA (1. mu.g/mL) for 15min to obtain cell culture supernatant, and histamine content in the supernatant was determined using a kit. The test result shows that the narcissus polysaccharide concentration of 100-250 mug/mL has a remarkable inhibition effect on the release of histamine from RBL-2H3 cells as shown in figure 5.
The preservative used in the embodiment of the invention is potassium sorbate, and a plurality of preservatives are available on the market at present and are not listed; the hydrolase inhibitors used in the embodiment of the invention are EDTA and sodium citrate, and have the common characteristic of certain chemical complexing capacity and many related chemicals, which are not listed; the crushing modes used in the embodiment of the invention include shredding, homogenate, extrusion, freeze-thaw crushing and the like, and the operation methods similar to the operation of crushing narcissus bulbs are many and are not listed.
Documents or patents cited in the present invention:
[1] chemical composition and biological activity study of Narcissus tazetta [ D ]. second university of military medical, 2013.
[2] Wangshi, Gaojian, Guwen, etc. the extraction and separation method of Chinese narcissus polysaccharide, CN105078872A [ P ]. 2015.
[3] Extraction, purification and detection of niweiqi polysaccharide and alkaloid [ D ]. Fujian agriculture and forestry university, 2013.
[4] The extraction process of crude polysaccharide in niweimin, pantomin, girl. narcissus bulbs is optimized [ J ] northern horticulture, 2012(10): 64-66.
While the foregoing description shows and describes the preferred embodiments of the present invention, it is to be understood that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as described herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (8)
1. The application of high-viscosity narcissus polysaccharide in preparing anti-allergy drugs is characterized in that the preparation method comprises the following steps:
(1) cleaning: taking fresh narcissus bulb, peeling off the outer skin, cutting off the root hair, and washing with clear water;
(2) crushing or homogenizing: pouring the cleaned narcissus bulb and an aqueous solution containing potassium sorbate and a metal ion complexing agent into a crusher for pulping;
(3) leaching: leaching at 0-50 ℃, supplementing an aqueous solution containing potassium sorbate and a metal ion complexing agent into the homogenate, and continuously stirring;
(4) coarse filtration: filtering the leaching solution with filter cloth;
(5) adjusting the pH value: adjusting the pH value to 2-6 by using acid, and uniformly stirring;
(6) centrifuging: respectively collecting supernatant and bottom precipitate;
(7) and (3) separating the lectin: freeze drying the precipitate to obtain crude lectin;
(8) concentration: concentrating the supernatant in vacuum;
(9) alcohol precipitation: precipitating the concentrated solution with ethanol, and rinsing the ethanol precipitate with ethanol water;
(10) and (3) drying: and (5) drying the alcohol precipitate in vacuum to obtain the narcissus polysaccharide.
2. The use of high viscosity narcissus polysaccharide as claimed in claim 1, wherein in step (3), the extraction time is 0.5-4 h.
3. The use of high viscosity narcissus polysaccharide in preparing anti-allergy medicine according to claim 1, wherein in steps (2) and (3), the addition amount of potassium sorbate is 0.05-1%.
4. The use of the high viscosity narcissus polysaccharide of claim 1 in preparing anti-allergic agent, wherein in steps (2) and (3), the metal ion complexing agent comprises edetate, citrate or phytic acid; the addition amount is 0.1-5%.
5. The use of the narcissus polysaccharide with high viscosity in the preparation of anti-allergy drugs according to claim 4, wherein the metal ion complexing agent is edetate disodium salt, and the addition amount is 0.1%.
6. The application of high-viscosity narcissus polysaccharide in preparing an anti-allergy medicament is characterized by comprising the following steps of:
(1) taking fresh narcissus bulb, peeling off the outer skin, cutting off the root hair, and washing with clear water;
(2) adding 200g of water hyacinth bulbs into 400mL of aqueous solution containing 0.05 percent of potassium sorbate and 0.1 percent of EDTA, and pulping by using a homogenizer;
(3) supplementing 1600mL of aqueous solution containing 0.05% of potassium sorbate and 0.1% of EDTA into the homogenate, and slowly stirring for 30 min;
(4) filtering with 200 mesh nylon cloth, and collecting filtrate;
(5) adjusting the pH value to 3-4 by trichloroacetic acid, and stirring for 30min at a speed of 15 r/min;
(6) centrifuging at 2000G for 30min, and collecting supernatant;
(7) adjusting the pH of the supernatant to about 6 with alkali, and concentrating the supernatant to 3-5 degrees brix by using a rotary evaporator;
(8) adding 3 times of alcohol into the concentrated solution, slowly stirring, standing for 60min, filtering with 100 mesh nylon cloth, collecting precipitate, and rinsing the precipitate with 70% alcohol for 2 times;
(9) the precipitate is dried in vacuum, and 8.21g of narcissus polysaccharide is obtained.
7. The application of high-viscosity narcissus polysaccharide in preparing an anti-allergy medicament is characterized by comprising the following steps of:
(1) taking fresh narcissus bulb, peeling off the outer skin, cutting off the root hair, and washing with clear water;
(2) shredding 2kg of water clematis bulbs, and putting into an extraction tank;
(3) adding 10kg of aqueous solution containing 0.05% potassium sorbate and 0.1% EDTA into the leaching tank, stirring for 120min, and discharging; leaching for 2 times again, and the solvent is 5kg each time;
(4) mixing the leaching solutions, filtering with 200 mesh nylon cloth, and collecting filtrate;
(5) adjusting the pH value to 3-4 by trichloroacetic acid, and stirring for 30min at a speed of 30 r/min;
(6) centrifuging by using a tubular centrifuge, and collecting a light phase;
(7) adjusting the pH value of the light phase to about 6 by using alkali, and concentrating the light phase to 3-5-degree brix by using a falling film evaporator;
(8) adding 3 times of alcohol into the concentrated solution, slowly stirring for 90min, filtering with 100 mesh nylon cloth, collecting precipitate, and rinsing the precipitate with 70% alcohol for 2 times;
(9) the precipitate is dried in vacuum, and 76.59g of narcissus polysaccharide is obtained.
8. The application of high-viscosity narcissus polysaccharide in preparing an anti-allergy medicament is characterized by comprising the following steps of:
(1) taking fresh narcissus bulb, peeling off the outer skin, cutting off the root hair, and washing with clear water;
(2) putting 100kg of water hyacinth bulbs into an extraction tank after passing through an extruder;
(3) adding 1000kg of aqueous solution containing 0.05% of potassium sorbate and 0.1% of EDTA into a leaching tank, stirring for 120min, and discharging; leaching for another time for 1 time, wherein the solvent is 800 kg;
(4) mixing the leaching liquor, filtering by using a filter bag with 200-400 meshes, and collecting filtrate;
(5) adjusting the pH value to 3-4 by using citric acid, and stirring for 30min at the speed of 60 r/min;
(6) centrifuging by using a tubular centrifuge, and collecting a light phase;
(7) adjusting the pH value of the light phase to about 6 by using alkali, and concentrating the light phase to 3-5-degree brix by using a falling film evaporator;
(8) adding 2.5 times of alcohol into the concentrated solution, slowly stirring for 120min, filtering with 100 mesh nylon cloth, collecting precipitate, and rinsing the precipitate with 75% alcohol for 2 times;
(9) the precipitate is dried in vacuum, namely 3.7kg of narcissus polysaccharide.
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| "Structure of Narcissus tazetta glucomannan";K.S.Zhauynbaeva;《CHEMISTRY OF NATURAL COMPOUNDS》;20030630;第39卷(第3期);第240-243页 * |
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